1. Technical Field
This invention relates to enzymatic amplification of DNA encoding a portion of a flagellin gene sequence in Borrelia, the portion being conserved among Borrelia species. More particularly, the invention relates to DNA oligomers complementary to portions of the flagellin gene of Borrelia burgdorferi which can be used to detect the organism, as well as other Borrelia species, in a sample.
2. Description of the Prior Art
Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the Sequence Listing. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which the invention pertains.
Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi (8, 32). The symptoms associated with the early stages of Lyme disease are rather general (eg. fever, malaise, etc.) and make diagnosis difficult (32). Furthermore, the immune response early in Lyme disease is unpredictable which renders antibody detection via ELISA methods of limited diagnostic value (10, 13). However, early detection and treatment reduces the probability of the more severe complications associated with later stages of Lyme disease (31).
One approach to the direct detection of microorganisms that is independent of host immune response is the relatively new technique known as the polymerase chain reaction or PCR (22). [See U.S. Pat. No. 4,683,195, issued Jul. 28, 1987; U.S. Pat. No. 4,683,202, issued Jul. 28, 1987; U.S. Pat. No. 4,800,159, issued Jan. 25, 1989; and U.S. Pat. No. 4,965,188, issued Oct. 23, 1990, each assigned to the Cetus Corporation, for a description of PCR technology. The contents of each of these patents are hereby incorporated by reference.] PCR involves the use of DNA sequence specific primers, a heat-stable DNA polymerase, and repeated temperature cycles of melting, hybridizing, and chain elongation to amplify specific gene sequences a millionfold in several hours. These amplified sequences can be detected in agarose gels or by using specific gene probes (27). Due to the degree of amplification achieved after PCR, detection of a single cell is possible under optimal conditions (23). Because of its sensitivity and specificity, the use of PCR has recently attracted much interest as a potential genetic approach to microbial diagnostics (1, 11, 20, 33).
Rosa and Schwan were the first to apply PCR to the detection of B. burgdorferi (26). Purified B. burgdorferi DNA was partially digested with a restriction endonuclease. The resultant restriction fragments were then inserted into plasmids and transformed into Escherichia coli. Competent clones were then screened, using hybridization techniques, as being "unique" to B. burgdorferi as tested against the closely related B. hermsii. The B. burgdorferi insert for a clone of interest was sequenced. Based upon this sequence, specific oligonucleotide primers were constructed for use in PCR. PCR with these primers was then performed on DNA from I8 strains of B. burgdorferi, B. hermsii, B. coriaceae, B. parkeri, B. turicatae. B. anserina, and B. crocidurae. Using this method, 17 of the 18 strains of B. burgdorferi were detected; no reactivity was associated with the other Borrelia species tested. Sensitivity to as few as five cells was reported with this assay.
Recently, the target for PCR amplification associated with detection of B. burgdorferi has been the outer surface protein (OSP) A gene (24, 25). This gene is located on a linear plasmid associated with the spirochete (4). In a study by Nielsen et al (24), oligonucleotide primers were constructed to the OSP A gene of the prototype strain B. burgdorferi B31 (ATCC-35210). Purified B. burgdorferi DNA was serially diluted and the number of cells estimated based upon the size of the B. burgdorferi genome. PCR was then conducted on the dilutions of B. burgdorferi DNA, B. hermsii, Treponema pallidum, T. denticola, Leptospira interrogans, and Staphylococcus aureus. Products were then identified specifically using a radioactively labeled oligonucleotide probe selected from the OSP A gene region between the two oligonucleotide primers. The assay was able to identify the B. burgdorferi DNA to a sensitivity of approximately fifty cells with the aid of hybridization. This assay did not identify any of the other organisms tested.
It has been reported in different studies that European and American strains differ in many ways, such as DNA homology, outer surface proteins, and plasmid composition (3, 6, 7, 17, 21, 29). In addition, it has been reported that European strains are more diverse than American strains (6). In other PCR-based assays for the detection of B. burgdorferi, some "unusual" strains were unable to be detected (25, 26). Therefore, one advantage of the subject invention is that it may offer versatility for the detection of many strains of B. burgdorferi.
There exists a continual need for efficient and sensitive ways to detect Borrelia burgdorferi in order to diagnose Lyme disease. The subject invention therefore provides for the use of PCR for the direct detection of B. burgdorferi. Furthermore, the subject invention provides a sensitive nonradioisotopic detection system coupled with PCR, by utilizing a biotinylated probe. The use of biotin as a label is disclosed in PCT International Application Publication No. WO 89/12063, published Dec. 14, 1989 (Mitchell and Merril), the contents of which is hereby incorporated by reference into this application.